Alistair Mathie

Guide to Receptors and Channels (GRAC), 5th edition: Guide to Receptors and Channels (GRAC), 5th edition

The Fifth Edition of the ‘Guide to Receptors and Channels’ is a compilation of the major pharmacological targets divided into seven sections: G protein‐coupled receptors, ligand‐gated ion channels, ion channels, catalytic receptors, nuclear receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside suggestions for further reading. Available alongside this publication is a portal at http://www.GuideToPharmacology.org which is produced in close association with NC‐IUPHAR and allows free online access to the information presented in the Fifth Edition.

Voltage-activated potassium channels in mammalian neurons and their block by novel pharmacological agents.

1. Electrophysiological studies have shown that a number of different types of potassium (K) channel currents exist in mammalian neurons. Among them are the voltage-gated K channel-currents which have been classified as fast-inactivating A-type currents (KA) and slowly inactivating delayed-rectifier type currents (KDR). 2. Two major molecular superfamilies of K channel have been identified; the KIR superfamily and the Shaker-related superfamily with a number of different pore-forming alpha-subunits in each superfamily. 3. Within the Shaker-related superfamily are the KV family, comprising of at least 18 different alpha-subunits that almost certainly underlie classically defined KA and KDR currents. However, the relationship between each of these cloned alpha-subunits and native voltage-gated K currents remains, for the most part, to be established. 4. Classical pharmacological blockers of voltage-gated K channels such as tetraethylammonium ions (TEA), 4-aminopyridine (4-AP), and certain toxins lack selectivity between different native channel currents and between different cloned K channel currents. 5. A number of other agents block neuronal voltage-gated K channels. All of these compounds are used primarily for other actions they possess. They include organic calcium (Ca) channel blockers, divalent and trivalent metal ions and certain calcium signalling agents such as caffeine. 6. A number of clinically active tricyclic compounds such as imipramine, amitriptyline, and chlorpromazine are also potent inhibitors of neuronal voltage-gated K channels. These compounds are weak bases and it appears that their uncharged form is required for activity. These compounds may provide a useful starting point for the rational design of novel selective K channel blocking agents.

Neuronal two-pore-domain potassium channels and their regulation by G protein-coupled receptors

Leak potassium currents in the nervous system are often carried through two‐pore‐domain potassium (K2P) channels. These channels are regulated by a number of different G protein‐coupled receptor (GPCR) pathways. The TASK subfamily of K2P channels are inhibited following activation of the G protein Gαq. The mechanism(s) that transduce this inhibition have yet to be established but there is evidence to support a role of phosphatidylinositol 4,5‐bisphosphate (PIP2) hydrolysis products, depletion of PIP2 itself from the membrane, or a direct action of activated Gαq on TASK channels. It seems possible that more than one pathway may act in parallel to transduce inhibition. By contrast, TRESK channels are stimulated following activation of Gαq. This is due to stimulation of the protein phosphatase, calcineurin, which dephosphorylates TRESK channels and enhances their activity. TREK channels are the most widely regulated of the K2P channel subfamilies being inhibited following activation of Gαq and Gαs but enhanced following activation of Gαi. The multiple pathways activated and the apparent promiscuous coupling of at least some K2P channel types to different G protein regulatory pathways suggests that the excitability of neurons that express K2P channels will be profoundly sensitive to variations in GPCR activity.

Activation of glutamate receptors and glutamate uptake in identified macroglial cells in rat cerebellar cultures.

1. Patch‐clamp methods have been used to examine the action of excitatory amino acids on three types of glial cell in cultures of rat cerebellum, namely type‐1‐like astrocytes, type‐2 astrocytes and oligodendrocytes. In addition we have examined glutamate sensitivity of the precursor cell (the O‐2A progenitor) that gives rise to type‐2 astrocytes and oligodendrocytes. 2. Glutamate (30 microM), quisqualate (3‐100 microM), (S)‐alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid (AMPA, 10‐30 microM) and kainate (10‐500 microM) were applied to cerebellar type‐2 astrocytes examined under whole‐cell voltage clamp. Each of these agonists induced inward currents in cells held at negative membrane potentials. The currents reversed direction near 0 mV holding potential. N‐Methyl‐D‐aspartate (NMDA, 30‐100 microM) or aspartate (30 microM) in the presence of glycine (1 microM) did not evoke any whole‐cell current changes in type‐2 astrocytes. 3. The distribution of glutamate receptors in type‐2 astrocytes was mapped with single‐ or double‐barrelled ionophoretic pipettes containing quisqualate or kainate. Application of these agonists (current pulses 100 ms, 50‐100 nA) to cells held at ‐60 mV evoked inward currents of 20‐120 pA in the cell soma and 10‐80 pA in the processes. Responses could also be obtained at the extremities of processes (approximately 60 microns from the soma). 4. Quisqualate or kainate (at 30 microM) applied to O‐2A progenitor cells from rat cerebellum or optic nerve induced whole‐cell currents (quisqualate 20‐30 pA; kainate 20‐50 pA, holding potential, Vh = ‐60 mV) that reversed near 0 mV. In common with type‐2 astrocytes, the progenitor cells did not respond to NMDA (30 microM). 5. Type‐1‐like astrocytes produced large inward currents to glutamate (30 microM). These currents remained inward‐going at holding potentials as positive as +80 mV and were not accompanied by any apparent noise increase. This result can be explained by the presence of an electrogenic glutamate uptake carrier. In cells kept up to 4 days in vitro, quisqualate, kainate and NMDA each failed to produce any whole‐cell current changes, indicating the absence of receptors in type‐1‐like astrocytes at this stage in culture. Furthermore the glutamate uptake currents in type‐1‐like astrocytes were inhibited when external Na+ was replaced by Li+, although Li+ was found to pass through the glutamate channel in type‐2 astrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Modifying the Subunit Composition of TASK Channels Alters the Modulation of a Leak Conductance in Cerebellar Granule Neurons

Two-pore domain potassium (K2P) channel expression is believed to underlie the developmental emergence of a potassium leak conductance [IK(SO)] in cerebellar granule neurons (CGNs), suggesting that K2P function is an important determinant of the input conductance and resting membrane potential. To investigate the role that different K2P channels may play in the regulation of CGN excitability, we generated a mouse lacking TASK-1, a K2P channel known to have high expression levels in CGNs. In situ hybridization and real-time PCR studies in wild-type and TASK-1 knock-outs (KOs) demonstrated that the expression of other K2P channels was unaltered in CGNs. TASK-1 knock-out mice were healthy and bred normally but exhibited compromised motor performance consistent with altered cerebellar function. Whole-cell recordings from adult cerebellar slice preparations revealed that the resting excitability of mature CGNs was no different in TASK-1 KO and littermate controls. However, the modulation of IK(SO) by extracellular Zn2+, ruthenium red, and H+ was altered. The IK(SO) recorded from TASK-1 knock-out CGNs was no longer sensitive to alkalization and was blocked by Zn2+ and ruthenium red. These results suggest that a TASK-1-containing channel population has been replaced by a homodimeric TASK-3 population in the TASK-1 knock-out. These data directly demonstrate that TASK-1 channels contribute to the properties of IK(SO) in adult CGNs. However, TASK channel subunit composition does not alter the resting excitability of CGNs but does influence sensitivity to endogenous modulators such as Zn2+ and H+.

Guide to receptors and channels, 2nd edition.

Guide to Receptors and Channels

Inhibition of the human two-pore domain potassium channel, TREK-1, by fluoxetine and its metabolite norfluoxetine.

1 Block of the human two‐pore domain potassium (2‐PK) channel TREK‐1 by fluoxetine (ProzacR) and its active metabolite, norfluoxetine, was investigated using whole‐cell patch‐clamp recording of currents through recombinant channels in tsA 201 cells. 2 Fluoxetine produced a concentration‐dependent inhibition of TREK‐1 current that was reversible on wash. The IC50 for block was 19 μM. Block by fluoxetine was voltage‐independent. Fluoxetine (100 μM) produced an 84% inhibition of TREK‐1 currents, but only a 31% block of currents through a related 2‐PK channel, TASK‐3. 3 Norfluoxetine was a more potent inhibitor of TREK‐1 currents with an IC50 of 9 μM. Block by norfluoxetine was also voltage‐independent. 4 Truncation of the C‐terminus of TREK‐1 (Δ89) resulted in a loss of channel function, which could be restored by intracellular acidification or the mutation E306A. The mutation E306A alone increased basal TREK‐1 current and resulted in a loss of the slow phase of TREK‐1 activation. 5 Progressive deletion of the C‐terminus of TREK‐1 had no effect on the inhibition of the channel by fluoxetine. The E306A mutation, on the other hand, reduced the magnitude of fluoxetine inhibition, with 100 μM producing only a 40% inhibition. 6 It is concluded that fluoxetine and norfluoxetine are potent inhibitors of TREK‐1. Block of TREK‐1 by fluoxetine may have important consequences when the drug is used clinically in the treatment of depression.

Characterization of the hyperpolarization-activated chloride current in dissociated rat sympathetic neurons.

1 Dissociated rat superior cervical ganglion (SCG) neurons have been shown to possess a hyperpolarization‐activated inwardly rectifying chloride current. The current was not altered by changes in external potassium concentration, replacing external cations with NMDG (N‐methyl‐D‐glucamine) or by addition of 10 mM caesium or barium ions. 2 The reversal potential of the current was altered by changing external anions. The anion selectivity of the current was Cl− > Br− > I− > cyclamate. All substituted permeant anions also blocked the current. 3 The current was blocked by DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid), 9AC (anthracene‐9‐carboxylic acid) and NPPB (5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid) but was unaffected by SITS (4‐acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulphonic acid) and niflumic acid. The effective blockers were voltage dependent; DIDS and NPPB were more effective at depolarized potentials while 9AC was more effective at hyperpolarized potentials. 4 The current was enhanced by extracellular acidification and reduced by extracellular alkalinization. Reducing external osmolarity was without effect in conventional whole‐cell recording but enhanced current amplitude in those perforated‐patch recordings where little current was evident in control external solution. 5 The current in SCG neurons was blocked by external cadmium and zinc. ClC‐2 chloride currents expressed in Xenopus oocytes were also sensitive to block by these divalent ions and by DIDS but the sensitivity of ClC‐2 to block by cadmium ions was lower than that of the current in SCG neurons. 6 Reverse transcriptase‐polymerase chain reaction (RT‐PCR) experiments showed the presence of mRNA for ClC‐2 in SCG neurons but not in rat cerebellar granule cells which do not possess a hyperpolarization‐activated Cl− current. 7 The data suggest that ClC‐2 may be functionally expressed in rat SCG neurons. This current may play a role in regulating the internal chloride concentration in these neurons and hence their response to activation of GABAA receptors.1. Dissociated rat superior cervical ganglion (SCG) neurons have been shown to possess a hyperpolarization‐activated inwardly rectifying chloride current. The current was not altered by changes in external potassium concentration, replacing external cations with NMDG (N‐methyl‐D‐glucamine) or by addition of 10 mM caesium or barium ions. 8 The reversal potential of the current was altered by changing external anions. The anion selectivity of the current was Cl− > Br− > I− > cyclamate. All substituted permeant anions also blocked the current.

Inhibition of N- and L-type calcium channels by muscarinic receptor activation in rat sympathetic neurons

Modulation of N- and L-type Ca2+ channels by oxotremorine-M (oxo-M) acting on muscarinic receptors and norepinephrine (NE) acting on alpha-adrenergic receptors was studied in superior cervical ganglion neurons. Oxo-M depresses dihydropyridine-augmented tail currents in whole-cell recordings, whereas NE does not. This modulation of L-type Ca2+ channels by oxo-M is abolished by adding 20 mM BAPTA to the pipette solution. Oxo-M, acting via a diffusible messenger, reduces the probability of opening of single N- and L-type channels recorded in cell-attached patches. We conclude that a diffusible messenger signaling pathway activated by oxo-M inhibits both N- and L-type Ca2+ channels, whereas a membrane-delimited pathway activated by oxo-M and NE inhibits only N-type Ca2+ channels.

Rectification of currents activated by nicotinic acetylcholine receptors in rat sympathetic ganglion neurones.

1. The inward rectification of the whole‐cell current evoked by acetylcholine (ACh) and other nicotinic agonists in rat sympathetic ganglion neurones has been studied using patch‐clamp recording techniques. The selective nicotinic agonist 1,1‐dimethyl‐4‐phenylpiperazinium iodide (DMPP) (20 microM) induced an average peak current of ‐367 pA at ‐50 mV but no detectable outward current at +50 mV. Similar observations were made with ACh and carbachol. 2. The current‐voltage relation of the whole‐cell response induced by DMPP was linear in the negative voltage range; however, there was no detectable outward current in the voltage range 0 to about +70 mV. Above +70 mV an outward current became clearly detectable. Rapid depolarizing jumps in the holding potential failed to reveal any rapidly decaying outward current. 3. The rectification was not alleviated by changing the main permeant cation, by removal of divalent cations from the intracellular or extracellular solutions or by altering the pH buffer in the extracellular solution from HEPES to Tris. 4. Intracellular magnesium ions can block the channel. This effect increases with depolarization, but dissociation outwards (i.e. permeation by Mg2+) appears to relieve the block at more extreme positive potentials. This effect alone, or in combination with the voltage dependence of the burst length, is unlikely to be able to account for the whole‐cell rectification in intact cells, much less that seen in cells perfused with Mg2(+)‐free intracellular medium. 5. When the reversal potential was shifted to approximately ‐50 mV (by the use of impermeant cations) nicotinic agonists produced small outward currents in the membrane potential range ‐20 to +10 mV while shifting it to about +40 mV produced small inward currents in the potential range 0 to +20 mV. The rectification therefore appears to be independent of the direction of current flow and is maximum at a potential positive to 0 mV. 6. At positive potentials the receptors desensitized much less than at negative potentials in the continued presence of agonist. Thus, exposure of the cells to a steady application of 30 microM‐ACh produced no detectable response if the cell was at a positive potential, but when the cell was stepped to a negative potential in the continued presence of ACh (at a time when much of the ACh current would be expected to have desensitized), ACh induced a large inward current. The onset of the ACh current had a time constant of 10 ms. It then decayed with a time constant of 790 ms as desensitization developed.(ABSTRACT TRUNCATED AT 400 WORDS)